Bacteria were grown overnight in TSB at 28 °C with shaking (200 rpm). TEM analysis was performed by the Laboratory of Electron Microscopy (Faculty of Biology, University of Gdansk, Poland). For the TEM analysis, bacteria were adsorbed onto carbon-coated grids (Sigma-Aldrich, Darmstadt, Germany), stained with 1.5% uranyl acetate, and directly examined with an electron microscope (Tecnai Spirit BioTWIN, FEI, New York, NY, USA) as described by Czajkowski et al. [111 (link)]. At least 10 photos were taken per strain to assess the morphology of Tn5 bacterial mutants.
Carbon coated grids
Manufactured by Merck Group
Sourced in Germany, United States, Sao Tome and Principe
Carbon-coated grids are a type of laboratory equipment used for sample preparation in various analytical techniques. They provide a stable and uniform surface for mounting and supporting samples, enabling effective analysis and imaging. The carbon coating enhances the conductivity and contrast of the samples, making them suitable for applications such as electron microscopy and other analytical methods that require thin, uniform sample supports.
Automatically generated - may contain errors
Lab products found in correlation
Tecnai spirit biotwin, by Thermo Fisher Scientific (1 mentions)
Uranyl acetate, by Merck Group (1 mentions)
Item software, by Thermo Fisher Scientific (1 mentions)
Leo 906, by Zeiss (1 mentions)
Goat anti mouse igg conjugated with 10 nm gold particles, by Merck Group (1 mentions)
Cm100, by Thermo Fisher Scientific (1 mentions)
5 protocols using carbon coated grids
1
Transmission Electron Microscopy of Tn5 Mutants
2
Transmission electron microscopy of phages
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For TEM analysis, 10 μl samples of purified phage were adsorbed onto carbon-coated grids (Sigma-Aldrich) for 3 min, stained with 1.5% uranyl acetate (Sigma-Aldrich) and examined using a Tecnai Spirit BioTWIN transmission electron microscope (FEI Company, Hillsboro, OR, USA). Images were collected using iTEM software (FEI Company). The visualization of phages was performed at the Laboratory of Electron Microscopy, Faculty of Biology, University of Gdansk, Gdansk, Poland.
3
Visualizing Baculovirus Proteins
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The carbon-coated grids (Sigma Aldrich, St Louis, MO) were floated on 10 µl of purified baculovirus solution for 30 min, and blocked with 5% bovine serum albumin (BSA) in PBS. After three times washing, the grids were exposed to mouse anti-His MAb (1:100 dilution) for 45 min. Subsequently, the grids were washed and exposed to goat anti-mouse IgG conjugated with 10 nm gold particles (1:50 dilution, Sigma, USA) for 30 min. After several washing steps, the grids were stained with 1% uranyl acetate and examined under the transmission electron microscope (Zeiss LEO 906, Germany).
4
Analyzing D. solani Tn5 Mutant Morphology
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To assess the morphology of D. solani Tn5 mutants, bacteria were grown overnight in TSB at 28°C with shaking (200 rpm). For the TEM analysis, bacteria were adsorbed onto carbon-coated grids (Sigma-Aldrich) stained with 1.5% uranyl acetate and directly examined with an electron microscope (Tecnai Spirit BioTWIN, FEI) as described previously (Czajkowski et al., 2015 (link), 2017b (link)). At least ten photos were taken per analyzed mutant and wild type strain.
5
Ultrastructural Analysis of D. solani
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To assess the morphology of D. solani cells treated with 50 μM SA, bacteria were grown overnight in TSB at 28 °C with shaking (200 rpm) in the presence of 50 μM SA. For the TEM analysis bacteria were adsorbed onto carbon-coated grids (Sigma) stained with 1.5 % uranyl acetate and directly examined with electron microscope (Philips CM100, EM, FEI Company) as described earlier (Szpitter et al. 2014) . The experiment was repeated independently one time with the same setup.
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